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Characterizing the control of nucleotide excision repair by ubiquitination through differential E1 phosphorylation

Reference	BB/E006590/1
Principal Investigator / Supervisor	Dr Thierry Nouspikel
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Institute for Cancer Studies
Funding type	Research
Value (£)	251,067
Status	Completed
Type	Research Grant
Start date	30/04/2007
End date	29/04/2010
Duration	36 months

Abstract

Nucleotide excision repair (NER), one of the most versatile DNA repair systems, is attenuated at the global genome level in terminally differentiated cells, whereas repair of active genes remains proficient. The NER deficiency in extracts of terminally differentiated macrophages can be complemented by the ubiquitin-activating enzyme E1, suggesting that a) NER is activated by ubiquitination, and b) the process is controlled at the level of the E1 enzyme. We have evidence that E1 phosphorylation is decreased in macrophages, compared with precursor cells, and we postulated that this prevents E1 from interacting with the E2 ubiquitin-conjugating enzyme used by NER. If this is the case, many other ubiquitination pathways may be regulated in such a way, given that there are only a few dozen E2 enzymes in our genome. Only 2 of the 4 phosphorylated serines in E1 are known, and we want to identify the others by mass spectrometry, or site-directed mutagenesis. We also plan to use site-directed mutagenesis to find which serine is de-phosphorylated in macrophages, and verify that such a point mutation prevents E1 from rescuing NER in macrophage extracts. We have indications that the NER enzyme activated by ubiquitination is TFIIH, a complex of 10 subunits. We plan to use 2D gel electrophoresis and mass spectrometry to identify the ubiquitinated subunit, and hopefully the target lysine to which ubiquitin is attached. We will also verify that ubiquitinated TFIIH can complement macrophage extracts, whereas non-ubiquitinated TFIIH cannot. Finally, we would like to identify other ubiquitination pathways potentially affected by the decrease in E1 phosphorylation observed in macrophages. We will supplement macrophage extracts with an excess of tagged ubiquitin, with or without addition of recombinant E1, isolate proteins bearing a tagged ubiquitin, and use mass spectrometry to identify those proteins that are only ubiquitinated upon addition of fully phosphorylated E1.

Summary

Our DNA is constantly damaged by environmental and internal factors, and needs to be continually repaired by several distinct repair systems. Nucleotide excision repair (NER) is probably the most versatile DNA repair system, and can handle UV-induced lesions, bulky chemical adducts, and intra-strand DNA crosslinks. Yet, this important repair system is largely non-functional in terminally differentiated cells, like brain cells or some white blood cells. The reason is probably that these cells never divide, and thus do not need to replicate their DNA. Provided that they can still repair the few genes they are using, which we have proved to be the case, these cells can dispense with repairing the bulk of their DNA. We are interested in understanding the mechanism of NER attenuation in terminally differentiated cells. We have determined that NER is activated by ubiquitination, and that this phenomenon does not occur in differentiated cells. Ubiquitination is a process carried on by a suite of three enzymes, E1, E2 and E3, in which a small protein called ubiquitin is attached to a target protein. This may trigger the degradation of the target or, as is the case for NER, may result in modifying its activity. NER is carried out by a dozen enzymatic complexes, totalizing about 30 different proteins subunits. We want to know which one is ubiquitinated, and prove that a lack of ubiquitination indeed impairs NER. Regulation of ubiquitination is generally controlled at the E3 level, as there are hundreds of E3 enzymes, allowing for differential ubiquitination of hundreds of targets. In the case of NER however, we have evidence that regulation occurs at the level of the unique E1 enzyme, probably by differential phosphorylation. Phosphorylation is yet another way to control the activity of an enzyme, by appending a small phosphate group to one of its amino acids. E1 is known to be phosphorylated, and it has been proposed that phosphorylation affects the way E1 interacts with the various E2 enzymes. We know that E1 comprises at least 4 phosphorylation sites, but the location of only 2 of these is known. We want to find the remaining two, determine which of the 4 sites is de-phosphorylated in differentiated cells, and prove that it results in a lack of ubiquitination of the NER enzyme involved. Finally, because there is only one E1 and a few dozen E2 enzymes, it is likely that such a regulatory mechanism will affect ubiquitination of many other targets than just NER. In fact, we postulated that it may be a clever strategy for cells that need to differentiate, to produce changes in many different pathways by modifying one 'master switch' enzyme. We want to find out if other ubiquitination pathways are indeed controlled by differential phosphorylation of E1, and to identify the cellular processes affected. NER is an extremely important DNA repair system, as exemplified by the highly cancer-prone disease xeroderma pigmentosum, in which an NER enzyme is deficient, resulting in hundreds of skin cancers. We have discovered a regulatory pathway that controls NER in healthy individuals, and we believe that a full understanding of this regulatory pathway may teach us a lot on how normal cells maintain the integrity of their DNA. In addition, we suspect that the precise regulatory mechanism that we have discovered may be used to control cellular differentiation, which is the process by which cells are committed to one function or another (e.g. become nerve cells versus muscle cells, etc.). We expect that our research will provide important information on how this complex process is controlled in normal individuals.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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